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PubMed1995

Glutathione transferases with novel active sites isolated by phage display from a library of random mutants.

Human glutathione transferase A1-1 can be expressed as a fusion protein with coat protein III of filamentous phage f1 in a form that allows selection among variant mutant forms based on specific adsorption to immobilized active-site ligands. A library of mutant enzymes differing in the active-site region was generated by random mutagenesis of ten amino acid residues involved in the binding of electrophilic substrates. Novel glutathione transferases with altered specificity for active-site ligands were isolated by adsorption of the fusion protein on the surface of phage to analogs of an electrophilic substrate. Thus, phage display of glutathione transferase affords a system for engineering novel binding specificities onto the pre-existing protein framework of the enzyme.

Amino Acid Sequence↗

PubMed1995

A simple protocol for identification of helical and mobile residues in membrane proteins.

A simple molecular dynamics (MD) simulations is protocol shown to predict whether a residue is in a structured alpha-helical segment or in a mobile loop or terminal segment of a membrane protein. The results are verified by comparisons with experimental NMR data. The protocol consists of performing several independent MD simulations on a polypeptide sequence of interest in a dielectric continuum with a relative permittivity epsilon = 2. The time histories of the individual angles between NH bond vectors at time 0 and time t later are calculated, and then Gaussian smoothing of typically 50 ps is applied. The smoothed data are subtracted from the original data to yield the short time fluctuations of the NH bond vectors, and then the rms deviations of the angles are calculated and compared to experimental NMR results. Pf1 coat protein and the c subunit of the E. coli F1F0 ATP synthase are used as examples of membrane proteins. The calculated NH bond rms fluctuations are in qualitative agreement with experimental NMR data in showing that each of these proteins has a mobile segment connecting two helices, as well as mobile N and C-terminal regions. This MD simulations protocol can demonstrate the presence of both the amphipathic and hydrophobic helices while hydropathy plots are able to detect only the hydrophobic helices present in membrane proteins.

Capsid↗

PubMed1996

Context dependence of mutational effects in a protein: the crystal structures of the V35I, I47V and V35I/I47V gene V protein core mutants.

The basis for the context dependence of the effects of core mutations on protein stability was investigated by comparing the structures of three gene V protein mutants with that of the wild-type protein. We previously examined a "swapped" mutant in which core residues Val35 and Ile47 were simply reversed so that the mutant had no hydrophobicity change from the native protein. The swapped mutant was destabilized by 3 kcal/mol per gene V protein dimer relative to the wild-type protein, demonstrating that factors other than hydrophobicity must make substantial contributions to the effects of mutations on the stability of the protein. Here we have determined the structure of this swapped mutant (V35I/I47V) as well as those of the two constituent mutants (V35I and I47V). We find that the structures of the mutant proteins are very similar to that of the wild-type protein except for the necessary addition or deletion of methylene groups and for slight positional shifts of atoms around each mutated residue. The structure of the double mutant is a composite of the structures of the two single mutants. In the mutant structures, the V35I mutation fills a cavity that exists in the wild-type protein and the I47V mutation creates a new cavity. The structures of the mutants indicate further that the reason the V35I and I47V mutations do not have opposite effects on stability is that the cavity in the wild-type protein filled by the V35I mutation is not optimally shaped for accommodating the additional methylene group of the isoleucine. These results support the concepts that the details of core packing have substantial influence on the effects of core mutations on protein stability and that these packing effects are major determinants of the context dependence of core mutation effects on stability.

Crystallography, X-Ray↗

PubMed1996

Subunit orientation in the filamentous virus Ff(fd, f1, M13).

The Ff filamentous viruses (fd, M13, f1) are important models for membrane protein assembly and are used extensively as cloning vectors and vehicles for peptide display. The thread-like virion (approximately 6 nm x 880 nm) comprises a single-stranded DNA genome sheathed by approximately 2700 copies of a 50-residue alpha-helical subunit (pVIII). The average inclination of pVIII subunits from the virion axis has been determined experimentally by polarized Raman microspectroscopy of oriented fd fibers, using the amide I Raman tensors obtained from a peptide single crystal. It is found that the average tilt angle, theta, between the pVIII alpha-helix and the virion axis falls within the range 13< theta <20 degrees.

Bacteriophage M13↗

PubMed1996

Role of capsid structure and membrane protein processing in determining the size and copy number of peptides displayed on the major coat protein of filamentous bacteriophage.

Filamentous bacteriophage virions can be engineered to display small foreign peptides in the N-terminal regions of all 2700 copies of the major coat protein (pVIII), but larger peptides can be accommodated only in hybrid virions, in which modified and wild-type coat protein subunits are interspersed. The copy number of peptides accepted in hybrid virions is generally believed to be related to peptide size: the larger the insert, the lower the number of modified coat protein subunits in the assembled virion. However, we show here that some large peptides can be displayed at a much higher copy number than smaller ones and that some relatively small peptides are poorly displayed, if at all, in hybrid virions. X-ray diffraction studies of a recombinant virion together with model building experiments with peptide and protein epitopes of known structure demonstrated that it is feasible to accommodate much larger structures, without perturbation of the capsid protein packing, than it has proved possible to generate in vivo. We show further that the insertion of certain peptides greatly slowed or even prevented the processing of the pVIII pro-coat by leader peptidase at the inner membrane of the Escherichia coli cell. A good correlation was found between the effect of the insert on the rate of the processing of the pro-coat, an essential step in virus assembly, and the number of the mature but modified proteins in the subsequently assembled hybrid virion. These results have important implications for the design of peptide display systems based on filamentous bacteriophage.

Amino Acid Sequence↗

PubMed1997

Filamentous phage IKe mRNAs conserve form and function despite divergence in regulatory elements.

As a means of determining whether there has been selection to conserve the basic pattern of filamentous phage mRNAs, the major mRNAs representing genes II to VIII have been defined for a phage distantly related to the Ff group specific for Escherichia coli hosts bearing F pili. Phage IKe has a genome with 55% identity with the Ff genome and infects E. coli strains bearing N pili. The results reveal a remarkably similar pattern of overlapping polycistronic mRNAs with a common 3' end and unique 5' ends. The IKe mRNAs, like the Ff phage mRNAs, represent a combination of primary transcripts and processed RNAs. However, examination of the sequences containing the RNA endpoint positions revealed that effectively the only highly conserved regulatory element is the rho-independent terminator that generates the common 3' end. Promoters and processing sites have not been maintained in identical positions, but frequently are placed so as to yield RNAs with similar coding function. By conserving the pattern of transcription and processing despite divergence in the regulatory elements and possibly the requirements for host, endoribonucleases, the results argue that the pattern is not simply fortuitous.

Base Sequence↗

PubMed1997

NMR structure of the principal neutralizing determinant of HIV-1 displayed in filamentous bacteriophage coat protein.

An NMR approach for structure determination of short peptides displayed on the surface of filamentous bacteriophage virions is demonstrated using the hexapeptide GPGRAF that constitutes the principal neutralizing determinant of HIV-1. This peptide was inserted near the N terminus of the major coat protein of bacteriophage fd. NMR studies of the recombinant protein solubilized in detergent micelles showed that the inserted peptide adopts a double bend S-shaped conformation that is similar to the antibody-bound structure determined by X-ray crystallography. This indicates that a peptide displayed on the bacteriophage coat protein has an enhanced propensity to adopt a conformation similar to that found in the native protein from which it is derived. This approach may be generally applicable to the structure determination of peptide epitopes and other small peptides.

Capsid↗

PubMed1997

Module swaps between related translocator proteins pIV(f1), pIV(IKe) and PulD: identification of a specificity domain.

In Gram-negative bacteria, type II and type III secretion and filamentous phage assembly systems use related outer membrane proteins for substrate-specific transport across the outer membrane. We show here that the specificity domain of the phage f1 outer membrane protein pIV is contained within the 149 N-terminal amino acid residues. When the pIV(f1) specificity domain is fused to the translocator domain of the related pIV of phage IKe, the chimeric construct supports f1 but not IKe assembly. Functional coupling between the two domains in this chimeric construct is poor and is improved by a single amino acid change in the translocator domain of the pIV(IKe). In native pIV(IKe), two amino acid changes within its specificity domain are both necessary and sufficient to change the specificity from IKe to f1 assembly. Analysis of 39 chimeric constructs between pIV(f1) and the outer membrane protein PulD of the pullulanase secretion system failed to identify a comparable exchangeable specificity domain. These results indicate that the two domains may not function autonomously, and suggest that tertiary and quarternary changes of the entire translocator component rather than of an autonomous functional domain are required for specific translocation across the outer membrane.

Amino Acid Sequence↗

PubMed1997

Selectively-infective phage (SIP): a mechanistic dissection of a novel in vivo selection for protein-ligand interactions.

Selectively-infective phage (SIP) is a novel methodology for the in vivo selection of interacting protein-ligand pairs. It consists of two components, (1) a phage particle made non-infective by replacing its N-terminal domains of geneIII protein (gIIIp) with a ligand-binding protein, and (2) an "adapter" molecule in which the ligand is linked to those N-terminal domains of gIIIp which are missing from the phage particle. Infectivity is restored when the displayed protein binds to the ligand and thereby attaches the missing N-terminal domains of gIIIp to the phage particle. Phage propagation is thus strictly dependent on the protein-ligand interaction. We have shown that the insertion of beta-lactamase into different positions of gIIIp, mimicking the insertion of a protein-ligand pair, led to highly infective phage particles. Any phages lacking the first N-terminal domain were not infective at all. In contrast, those lacking only the second N-terminal domain showed low infectivity irrespective of the presence or absence of the F-pilus on the recipient cell, which could be enhanced by addition of calcium. An anti-fluorescein scFv antibody and its antigen fluorescein were examined as a protein-ligand model system for SIP experiments. Adapter molecules, synthesized by chemical coupling of fluorescein to the purified N-terminal domains, were mixed with non-infective anti-fluorescein scFv-displaying phages. Infection events were strictly dependent on fluorescein being coupled to the N-terminal domains and showed a strong dependence on the adapter concentration. Up to 10(6) antigen-specific events could be obtained from 10(10) input phages, compared to only one antigen-independent event. Since no separation of binders and non-binders is necessary, SIP is promising as a rapid procedure to select for high affinity interactions.

Calcium Chloride↗

PubMed1997

fd coat protein structure in membrane environments: structural dynamics of the loop between the hydrophobic trans-membrane helix and the amphipathic in-plane helix.

By performing multidimensional solution NMR experiments on micelle samples it was possible to determine the structure of the membrane-bound form of fd coat protein based on short-range distance and dihedral angle constraints using distance geometry and simulated annealing calculations. Its dynamics were described by 15N relaxation measurements (T1, T2, heteronuclear nuclear Overhauser enhancement (NOE)) fitted with the Lipari-Szabo model-free formalism adapted for the transmembrane and in-plane helices of a membrane protein. The overall correlation time of the protein in micelles was found to be approximately 9 ns, and the local motion of each backbone N-H vector was described by an order parameter and an effective correlation time. The 50 residue protein has an amphipathic alpha-helix (residues 7 to 16) and a hydrophobic alpha-helix (residues 27 to 44), which were found to be approximately perpendicular on the basis of NOEs in the residues that connect the two helices. The residues connecting the helices are of particular interest in membrane proteins, and in this case the loop consists of two turns. The relaxation data show the presence of an extra motion in the amphipathic alpha-helix on the nanosecond timescale and additional flexibility of several residues in the loop connecting the two helices.

Amino Acid Sequence↗

PubMed1998

Equilibrium and kinetic binding analysis of the N-terminal domain of the Pf1 gene 5 protein and its interaction with single-stranded DNA.

The Pf1 gene 5 protein is a single-stranded DNA-binding protein that binds cooperatively to the viral strand of Pf1 DNA during replication. A variety of N-terminal fragments of the Pf1 gene 5 protein have been expressed and purified. We have identified an N-terminal single-stranded DNA-binding domain (residues 1 to 105) that is much more globular than the intact protein (1 to 144). Larger fragments (1 to 115) as well as smaller fragments (1 to 91) were unable to bind DNA effectively. Analysis of the truncated proteins by gel retardation and fluorescence anisotropy indicates that the N-terminal domain binds DNA with a reduced affinity, due principally to a reduction in cooperativity, and that binding is highly concentration-dependent. Kinetic analysis shows that the rates of association and dissociation of the N-terminal domain from the complex with DNA are faster than those observed for the intact protein. The results suggest that the flexible C-terminal domain of the Pf1 gene 5 protein plays an important role in protein-protein interactions that stabilise adjacent protein dimers in the DNA-protein complex.

Biosensing Techniques↗

PubMed1998

Roles of pIII in filamentous phage assembly.

Filamentous phage protein III (pIII), located at one end of the phage, is required for infectivity and stability of the particle. Cells infected with phage from which gene III has been completely deleted produce particles that are not released into the medium but stay associated at the surface. These particles are much longer than normal phage. They can be released by subsequent expression of pIII. Viewed with the electron microscope, cells infected with gene III deletion phage are decorated with structures that resemble extremely long pili. Surprisingly, such cells are viable and can form colonies. The pIII deficiency can be complemented in trans, but there is a threshold concentration below which assembly does not occur. Above this threshold, pIII is used very efficiently and is incorporated into infectious but longer than unit length phage. As the concentration of pIII is increased, the number of infectious particles increases, and their average length decreases.pIII stabilizes pVI, a second phage protein found at the pIII end of the particle. In the absence of pIII, degradation of pVI is very rapid. pIII is thus not only required for infectivity and particle stability, but to terminate assembly and release the phage from its assembly site.

Capsid Proteins↗

PubMed1999

Effects of temperature and Y21M mutation on conformational heterogeneity of the major coat protein (pVIII) of filamentous bacteriophage fd.

Solid-state NMR spectroscopy was used to analyze the conformational heterogeneity of the major coat protein (pVIII) of filamentous bacteriophage fd. Both one and two-dimensional solid-state NMR spectra of magnetically aligned samples of fd bacteriophage reveal that an increase in temperature and a single site substitution (Tyr21 to Met, Y21M) reduce the conformational heterogeneity observed throughout wild-type pVIII. The NMR results are consistent with previous studies indicating that conformational flexibility in the hinge-bend segment that links the amphipathic and hydrophobic helices in the membrane-bound form of the protein plays an essential role during phage assembly, which involves a major change in the tertiary, but not secondary, structure of the coat protein.

Capsid↗

PubMed1999

Isolation of chloroform-resistant mutants of filamentous phage: localization in models of phage structure.

Interaction of fd or M13 filamentous phage with a chloroform/water interface induces morphological change, contracting the filaments sequentially into shortened rods (I-forms), and then into spheroidal particles (S-forms). To further investigate this phage contraction, 34 and 26 chloroform-resistant isolates of fd and M13, respectively, were selected after chloroform treatment of wild-type phages at pH 8. 2 and 4 degrees C. DNA sequencing of gene VIII of the 34 fd isolates revealed five different mutants: these were D5H, M28L, V31L, I37T, and S50T. All 26 M13 isolates were I37T. These mutants exhibited variable sensitivity to chloroform, but all contracted much more slowly than wild-type phage during treatment at 4 degrees C. They all contracted like wild-type phage at 37 degrees C. Site-directed mutagenesis showed that the indicated single mutations carried the chloroform resistance. In structural models of the phage, the D5H locus is on the outside and the S50T locus is on the inside. The M28L and I37T loci are buried in a mostly hydrophobic region in the middle. Although these four mutants are spread out radially, they are localized in the axial direction into a thin disk in the model. The last mutant locus, V31L, is out of this disk, but this locus is proximal to the M28L and I37T loci and also in contact with the surface via a deep hydrophobic hole or depression. These five mutants, their locations, and their variable affects on contraction suggest that chloroform-induced contraction involves a specific mechanism rather than a generalized solvent-induced denaturation and that the critical structural changes occur in a localized level in the phage. These results add weight to suggestions that the sequential contraction of filaments-->I-forms-->S-forms mimic corresponding steps in phage penetration, and, in the reverse order, for phage assembly.

Bacteriophage M13↗

PubMed1999

Functionally important correlated motions in the single-stranded DNA-binding protein encoded by filamentous phage Pf3.

To elucidate the interplay between different parts of dimeric single-stranded DNA-binding proteins we have studied the correlated motions in the protein encoded by filamentous phage Pf3 via the combined use of 15N-NMR relaxation experiments, molecular dynamics simulations and essential dynamics calculations. These studies provide insight into the mechanism underlying the protein-DNA binding reaction. The most important motions can be described by a few essential modes. Most outstanding is the correlated symmetric motion of the DNA-binding wings, which are far apart in the structure. This motion determines the access of DNA to the DNA-binding domain. A correlation between the motion of the DNA-binding wing and the complex loop is indicated to play a role in the cooperative binding of the protein to DNA. These motions are in the nanosecond regime in correspondence with the 15N-NMR relaxation experiments.

DNA, Single-Stranded↗

PubMed1999

Epitope structures recognised by antibodies against the major coat protein (g8p) of filamentous bacteriophage fd (Inoviridae).

To map the accessible surface of filamentous bacteriophage fd particles, the epitope structures of polyclonal rabbit serum and three mouse monoclonal antibodies raised against complete phage were analysed. Western blot analysis confirmed the major coat protein, gene VIII product (g8p or pVIII), to be the antigen. Overlapping peptides were synthesised by spot synthesis on cellulose membranes, covering the whole sequence of g8p. Each of the three tested monoclonal antibodies, B62-FE2, B62-GF3/G12 and B62-EA11, reacted with a core epitope covering ten amino acid residues at or near the amino terminus of g8p. The epitope recognised by B62-FE2 consists of the ten N-terminal amino acid residues of g8p. Extension of the amino terminus by various sequences did not inhibit binding, indicating that a terminal amino group is not essential for the interaction. Both B62-GF3/G12 and B62-EA11 recognise internal epitopes covering amino acid residues 3 to 12 of g8p. The epitopes of the polyclonal rabbit serum were also confined to the 12 N-terminal amino acid residues. The contribution of individual amino acid residues to the binding was analysed by a set of peptides containing individual amino acids exchanged by glycine. Accessible residues were Glu2, Asp4, Asp5, Pro6, Lys8, Phe11 and Asp12. The positions of the essential amino acid residues within the epitope are in accordance with a helical conformation of the amino-terminal region of g8p. Further, the results suggest new designs of phage display screening vectors to improve their performance in analysing non-linear epitopes.

Amino Acid Sequence↗

PubMed1999

Crystal structure of the two N-terminal domains of g3p from filamentous phage fd at 1.9 A: evidence for conformational lability.

Infection of Escherichia coli by filamentous bacteriophages is mediated by the minor phage coat protein g3p and involves two distinct cellular receptors, the F' pilus and the periplasmic protein TolA. Recently we have shown that the two receptors are contacted in a sequential manner, such that binding of TolA by the N-terminal domain g3p-D1 is conditional on a primary interaction of the second g3p domain D2 with the F' pilus. In order to better understand this process, we have solved the crystal structure of the g3p-D1D2 fragment (residues 2-217) from filamentous phage fd to 1.9 A resolution and compared it to the recently published structure of the same fragment from the related Ff phage M13. While the structure of individual domains D1 and D2 of the two phages are very similar (rms<0.7 A), there is comparatively poor agreement for the overall D1D2 structure (rms>1.2 A). This is due to an apparent movement of domain D2 with respect to D1, which results in a widening of the inter-domain groove compared to the structure of the homologous M13 protein. The movement of D2 can be described as a rigid-body rotation around a hinge located at the end of a short anti-parallel beta-sheet connecting domains D1 and D2. Structural flexibility of at least parts of the D1D2 structure was also suggested by studying the thermal unfolding of g3p: the TolA binding site on D1, while fully blocked by D2 at 37 degrees C, becomes accessible after incubation at temperatures as low as 45 degrees C. Our results support a model for the early steps of phage infection whereby exposure of the coreceptor binding site on D1 is facilitated by a conformational change in the D1D2 structure, which in vivo is induced by binding to the F' pilus on the host cell and which can be mimicked in vitro by thermal unfolding.

Amino Acid Sequence↗

PubMed1999

Inhibition of cytoplasmic antigen, glucose- 6-phosphate dehydrogenase, by VH-CH1, an intracellular Fd fragment antibody derived from a semisynthetic Fd fragment phage display library.

A library of Fd fragment antibody binding proteins was created by random mutation of 15 nucleotides within the CDRIII region of the immunoglobulin heavy chain gene and displayed as Fd coat protein fusion constructs of M13 phage. The library was screened for those VHbinding sites that bound glucose-6-phosphate dehydrogenase (G6PD). One isolate (DH27bp) inhibited G6PD activity by 85 %. The DH27bpgene was re-engineered, placed in a eukaryotic expression vector having an isopropyl-beta-delta-thiogalactopyranoside (IPTG) inducible promoter, and transfected and then expressed in Chinese hamster V79 cells. G6PD activity was completely inhibited. Removal of IPTG reverted the cell to full G6PD activity. The intracellular dynamics of the G6PD/DH27bpcomplex showed that when the proteasomes of cells expressing DH27bpwere inhibited (N -acetyl-Leu-Leu-norleucinal or lactacystin) G6PD activity increased. Metabolic labelling of newly synthesized IPTG-induced proteins during/absence of proteasomal inhibitors showed that both G6PD and DH27bpare signaled for degradation when the intracellular complex is formed. Furthermore, semi-quantitative RT/PCR demonstrated that G6PD mRNA is upregulated over the time course of G6PD inactivation by DH27bpFd binding protein. These effects were not observed in those cells expressing a non-mutated Fd (UMHC) or in IPTG-treated non-transduced V79 cells. Our results demonstrate that an Fd-based intracellular binding protein can find and disable the function of a specific intracellular target and once the Fd expression is repressed the activity of intracellular targeted protein can revert to normal.

Acetylcysteine↗